Targeted proteomics identify metabolism-dependent interactors of yeast cytochrome c peroxidase: implications in stress response and heme trafficking

Abstract

Recently we discovered that cytochrome c peroxidase (Ccp1) functions primarily as a mitochondrial H₂O₂ sensor and heme donor in yeast cells. When cells switch their metabolism from fermentation to respiration mitochondrial H₂O₂ levels spike, and overoxidation of its polypeptide labilizes Ccp1's heme. A large pool of heme-free Ccp1 exits the mitochondria and enters the nucleus and vacuole. To gain greater insight into the mechanisms of Ccp1's H₂O₂-sensing and heme-donor functions during the cell's different metabolic states, here we use glutathione-S-transferase (GST) pulldown assays, combined with 1D gel electrophoresis and mass spectrometry to probe for interactors of apo- and holoCcp1 in extracts from 1 d fermenting and 7 d stationary-phase respiring yeast. We identified Ccp1's peroxidase cosubstrate Cyc1 and 28 novel interactors of GST-apoCcp1 and GST-holoCcp1 including mitochondrial superoxide dismutase 2 (Sod2) and cytosolic Sod1, the mitochondrial transporter Pet9, the three yeast isoforms of glyceraldehyde-3-phosphate dehydrogenase (Tdh3/2/1), heat shock proteins including Hsp90 and Hsp70, and the main peroxiredoxin in yeast (Tsa1) as well as its cosubstrate, thioreoxin (Trx1). These new interactors expand the scope of Ccp1's possible roles in stress response and in heme trafficking and suggest several new lines of investigation. Furthermore, our targeted proteomics analysis underscores the limitations of large-scale interactome studies that found only 4 of the 30 Ccp1 interactors isolated here.